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What Is the Correlation Between Corrected Optical Density and Cell Number?

Optical density -- a measure of the extent to which a substance transmits light -- is often used to measure the concentration of cells or particles that are dispersed, but not dissolved, in a fluid. The corrected optical density of this type of mixture, known as a suspension, is directly proportional to the concentration, density or number of cells dispersed in it.

Beer-Lambert Law
- The Beer-Lambert law, which states that absorbance is proportional to or has a linear relationship with, the concentration of the absorbing species, is only true for optical densities below 0.4. However, samples with optical densities higher than 0.4 can be diluted by a defined dilution factor and their optical densities measured. If the measured values are divided by the dilution factor, to produce the corrected optical density, this new value is proportional to the number of cells in the specimen.
Spectrophotometer
- Optical density is measured by an instrument, known as a spectrophotometer, which compares the brightness of light emitted from two sources, one of which has certain standard characteristics. As light passes through a cell suspension, the light is scattered by the cells so that less of it reaches a photoelectric cell -- an electronic device whose electrical output varies according to the strength of the light falling on it -- inside the spectrophotometer. Thus, the higher the number of cells in the suspension the weaker the electrical output of the photoelectric cell and vice versa.
Optical Density Correction
- At optical densities above 0.4, the degrees to which cells can be shadowed by other cells or reflect light many times is appreciable. At higher optical densities, the uncorrected optical density of cell suspensions is no longer directly proportional to cell number. However, the uncorrected optical density of dilute cell suspensions is almost proportional to their accurately known relative densities -- the ratio of their densities to that of a reference substance, such as water -- so it is a reasonable working assumption that the uncorrected optical density of a highly dilute cell suspension is, in fact, its true optical density.
Relative Optical Density
- By multiplying the relative density value of any cell suspension by the basic optical density value obtained from the highly dilute cell suspension, it is possible to obtain the corrected optical density. However, to make the measurement as accurate as possible, the basic optical density value should be obtained from multiple spectrophotometer readings.

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